The present invention relates to the field of transgenic plants. Specifically, the invention provides compositions and methods for the transformation of plastids in plants from the Cruciferae family.
Several publications are parenthetically referenced in this application in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference in the present specification as though set forth herein in full.
The plastid genome of higher plants is a circular double-stranded DNA molecule of 120-160 kb which may be present in 1,900-50,000 copies per leaf cell (Palmer, 1991; Bendich, 1987). Stable transformation of the tobacco plastid genome (plastome) has been achieved through the following steps: (i) introduction of transforming DNA, encoding antibiotic resistance, by the biolistic process (Svab et al. 1990a; Svab and Maliga 1993) or PEG treatment (Golds et al. 1993; O""Neill et al., 1993), (ii) integration of the transforming DNA by two homologous recombination events and (iii) selective elimination of the wild-type genome copies during repeated cell divisions on a selective medium. Spectinomycin resistance has been used as a selective marker encoded either in mutant plastid 16S ribosomal RNA genes (Svab et al. 1990a; Staub and Maliga 1992; Golds et al. 1993), or conferred by the expression of an engineered bacterial aadA gene (Svab and Maliga 1993). Vectors which utilize aadA as a selectable marker gene and target the insertion of chimeric genes into the repeated region of tobacco plastid genome are available (Zoubenko et al., 1994). Selection of plastid transformants by kanamycin resistance, based on the expression of neomycin phosphotransferase (kan gene), is more difficult but also feasible (Carrer et al., 1993; Carrer and Maliga, 1995).
To date, stable plastid transformation in higher plants has been reported in tobacco only (reviewed in Maliga, 1993; Maliga et al., 1993). Transplastomic plants from other agriculturally and pharmaceutically important species are highly desirable. Expression of foreign genes of interest in the plastids of higher plants in the family Cruciferae provides several advantages over nuclear expression of foreign genes. These are 1) expression of exogenous DNA sequences in plastids eliminates the possibility of pollen transmission of transforming DNA; 2) high levels of protein expression are attainable; 3) the simultaneous expression of multiple genes as a polycistronic unit is feasible and 4) positional effects and gene silencing which may result following nuclear transformation are also eliminated.
The present invention provides improved methods for the generation of stably transformed, transplastomic plants. In one embodiment of the invention, cotyledon cells are cultured in high auxin liquid medium for a sufficient time period to stimulate uniform cell division. Initial culture is at a high density (50-200 cotyledons/20 ml). The cotyledons are then transferred to agar-solidified medium for delivery of exogenous, transforming DNA. Following delivery of transforming DNA, the cotyledons are transferred at a lower density (25-30/50 ml) to a medium containing high cytokinin level and the selection agent to facilitate selection of transformants and plant regeneration. Presence of the exogenous DNA in the plastid genome is then confirmed by Southern blot analysis or PCR.
The transforming DNA molecules of the invention have several distinct features. These are 1) targeting segments flanking the foreign gene of interest consisting of plastid DNA sequences from the plant to be transformed, thereby facilitating homologous recombination of the transforming DNA into a pre-determined region of the plastid genome; 2) a selectable marker gene disposed within the targeting segment, conferring resistance to a selection agent; 3) 5xe2x80x2 and 3xe2x80x2 regulatory sequences derived from plastid DNA operably linked to sequences encoding a foreign gene of interest thereby enhancing expression of the transforming DNA and stability of encoded mRNA; and 4) at least one cloning site adjacent to the selectable marker gene for insertion of the foreign gene of interest which by itself is not selectable. Since the selectable marker gene and the foreign gene of interest form a heterologous block of contiguous sequence, integration of both genes into the plasid genome is effected.
In another embodiment of the invention, leaf cells are initially treated with high auxin media, followed by transformation with the transforming DNA and culturing in the presence of high cytokinin levels and a predetermined selection agent. Cells containing transformed plastids are maintained in the presence of the selection agent facilitating the obtention of homoplasmic cells which can then be regenerated into transplastomic plants.
Thus, the present invention provides novel methods and compositions for creating transplastomic plants. The genus Arabidopsis belongs to the mustard or crucifer family (Brassicaceae or Cruciferae), a widely distributed family of approximately 340 genera and 3350 species. The family is of significant economic importance as a source of vegetable crops, oil seeds, spices and, to a lesser extent, ornamentals. Much of its agricultural importance derives from the genus Brassica. Examples for Brassica ssp. of economic importance are: Brassica napus (oil seed), Brassica juncea (oil seed), Brassica campestris (oil seed), Brassica juncea (oil seed), Brassica oleracea (broccoli, cauliflower, cabbage) Brassica nigra (black mustard) and Brassica hirta (white mustard).
Plastid transformation in Arabidopsis thaliana a model species for plant research (Meyerowitz and Sommerville, 1994) and Brassica ssp., an important agricultural crop is exemplified herein. These methods are suitable for transformation of plastids in other plants from the Cruciferae family.